IE910336A1 - Preparation and use of gene banks of synthetic human antibodies ("synthetic human-antibody libraries") - Google Patents

Abstract

Synthetic human antibody libraries are constructed by'generation of random sequences for the hypervariable regions under the restrictions (a) or (e), batch A or batch B according to Table 1 and subsequent insertion into an expression vector' (sic). Synthetic human antibody libraries can be produced by using almost randomly synthesised oligonucleotides coding for each of the three hypervariable regions in the variable parts of the heavy and light chains (regions CDR1, CDR2 and CDR3).

Description

« BEHRINGWERKE AKTIENGESELLSCHAFT HOE 90/B 006J - Ma 834 Dr. Lp/rd Preparation and use of gene banks of synthetic human 5 antibodies (synthetic human-antibody libraries) The invention relates to the preparation and use of gene banks of synthetic human antibodies (huAb) or parts of antibodies which contain the antigen-binding domain.

Starting from a huAb framework in a suitable vector, the hypervariable regions of the antibody cDNA are formed by almost randomly combined oligonucleotides. Relatively conserved amino acids in the hypervariable regions have here been taken account of in the choice of appropriate nucleotides during the oligonucleotide synthesis and the ratio of the nucleotides used is likewise chosen such that a nonsense codon is to be expected at most in every 89th position. Expression of this synthetic huAb cDNA in microbial expression systems, e.g. in E. coli in the vector pFMT which is described below, thus makes a synthetic huAb library with a comprehensive repertoire for screening using selected antigens available in vitro.

The human or mammalian immune system comprises an estimated number of between 106 and 10® different antibodies.

This number of antibodies seems to be sufficient to cause an immune reaction of the body both against all naturally occurring antigens and against artificial antigens. If it is furthermore taken into account that often different antibodies react with the same antigen, the repertoire of antibodies that are really different would rather be in the region from 106 to 107.

Up to now specific antibodies have always been obtained starting from an immunization with the particular antigen, for example injection of the antigen into the body or in vitro incubation of spleen cells with this antigen.

In the case of polyclonal antibodies, the immunoglobulins IE 91336 - 2 , can then be isolated from the serum and the specific antibodies can be isolated therefrom, e.g. by absorption methods. Monoclonal antibodies are isolated from the cell supernatants or from the cell lysate of spleen tumor cells (hybridoma cells) which have been fused with individual B lymphocytes and cloned. The abovementioned methods are unsuitable in particular for the preparation of specific human antibodies or human monoclonal antibodies .

The present invention therefore has the object of developing a generally usable method for generating specific human monoclonal antibodies (huMAbs) or parts of antibodies, which contain synthetic hypervariable domains.

It has now been found that by using almost randomly synthesized oligonucleotides coding for the three hypervariable regions of each variable part of heavy or light chains (called CDR1, 2 and 3, CDR meaning complementary determining region) synthetic human gene banks can be generated. The synthesized antibody DNA was then preferably ligated into an antibody expression vector especially constructed for this purpose, namely the vector pFMT, preferably after amplification using the polymerase chain reaction (PCR).

The oligonucleotides which are used for the synthesis of the variable domains of heavy and light chains are compiled in Tab. 1. Set A here contains fewer limitations than set B. The limitations below the synthesis of the hypervariable regions (see CDR regions in Tab. 4) being random were made in H3, H4, H6, L2, L3 and L5 in set A in order firstly to allow for positions in the sequence for certain conserved amino acids, secondly to reduce the number of stop codons, and thirdly to incorporate a new restriction site. (a) In order to reduce the probability of the stop codon IE 91336 - 3 occurring, only half the amount of the three other nucleotides was allowed for T at the first position of each codon and A was omitted at the third position of each codon, in each case. As a statistical average, only every 89th codon will thus be a stop codon. (b) For the 2nd codon in the CDR1 region of the light chain, only those nucleotides were allowed which code for the amino acids V, A or G. (c) Likewise, only those combinations coding for V, I or M were allowed for codon No. 10 in the CDR1 region of the light chain and for codon No. 4 in the CDR1 region of the heavy chains. (d) In the CDR3 region of the light chain, only those 15 nucleotides coding for the amino acid glutamine were allowed for codon No. 1. (e) In the CDR2 region of the heavy chain, only those nucleotides coding for the amino acid tyrosine were allowed for codon No. 11. (f) An A was advantageously incorporated at the third position of the last codon in the CDR2 region of the heavy chain in order to introduce a restriction site for Mlul.

The random nature of these oligonucleotides was prefer25 ably limited even further in those positions where predominantly one or few amino acids occur (set B in Tab. 1, the limitations here are based on the tables by Rabat et al. (1987), Sequences of Proteins of Immunological Interest-U.S. Dept. of Health and Human Services, U.S.

Government Printing Offices). A list of the corresponding nucleotides and brief notes on the codon combinations are compiled in Tab. 1 and in the notes for Tab. 1.

IE 91336 - 4 , After ligation of equimolar amounts of the oligonucleotides Hl to H7 and LI to L5, these are ligated into the pretreated expression vector pFMT. Preferably, a PCR step using the primers Hl and H8, or LI and L6 should be carried out beforehand in order to amplify the amount of DNA. After producing suitable restriction sites at the ends of the antibody DNA using appropriate restriction enzymes, the DNA is ligated into the antibody expression vector pFMT in the same manner as above (see examples).

The expression pFMT makes possible the expression of antibody cDNA and the subsequent secretion of the expression products in bacteria (E.coli). The antibody operon of the plasmid contains the sequences of the variable parts of both the heavy and light chain of an antibody.

Suitable leader sequences from the amino terminal part of a bacterial protein makes secretion of the antibody parts possible. The leader sequences are cleaved off by a bacterial enzyme during the secretion. During the secretion of the antibody cDNA products, the light and heavy chains of the antibody (with or without an adjacent constant domain) become associated. This results in the formation of an antibody or antibody fragment which, in either case, contains a functional antigen binding site. Similar constructs for individual antibodies have also been described by other authors (Better et al. (1988), Science 240, 1041, and Skerras & Pliickthun (1988), Science 240, 1038).

In the synthetic human-antibody library formed by the expression in, for example, E. coli, the desired human antibodies or antibody parts are found by screening bacterial clones using the selected antigen. In a preferred embodiment, an additional sequence which codes for a marker peptide, for example a TAG sequence, is incorporated so that the expression products can be detected in a simple way using established monoclonal antibodies against the marker peptide (Wehland et al. (1984), EMBO J. 3, 1295).

IE 91336 , The abovementioned exemplary formulations and the examples below shall be understood as illustrating but not restricting the invention.

The invention therefore relates to gene banks of syn5 thetic huAb or antigen-binding parts thereof, obtained by means of (1) cDNA for the hypervariable regions generated on a random basis, where the random sequences are limited by clauses (a) to (e) set A or in accordance with Tab. 1, set B, (2) preferably a subsequent amplification step of these random sequences and (3) ligation of the said cDNA into a suitable expression vector, preferably pFMT, an additional coding sequence for a marker peptide being incorporated in a preferred embodiment.

The invention also relates to a process for the separation of the abovementioned gene banks, and the process and the use thereof for the isolation of clones which secrete specific antibodies or antigen-binding parts thereof.

Finally, the invention is explained in detail in the examples and contained in the patent claims.

Examples: Example 1: Preparation of an antibody expression vector The plasmid pKK233-2 (Amann and Brosius, (1985) Gene 40, and Straus and Gilbert (1985) Proc. Natl. Acad. Sci. 82, 2014) was chosen as base vector for the construction of the antibody expression vector (Fig. 1).

Before the incorporation of the antibody operon, the plasmid was cut with Sail and BamHI, the ends were filled in with Klenow polymerase and ligated. By doing so, the two restriction sites and the DNA between them were IE 91336 - 6 deleted.

Additionally, the plasmid was cleaved with Hindlll, the ends were filled in with Klenow polymerase and ligated using BamHI linkers. By this procedure, the Hindlll restriction site was removed and a BamHI site inserted. The antibody DNA was inserted into this modified plasma. A simplified structure of the antibody operon coding for a dicistronic antibody mRNA is shown in Tab. 2. In order to make possible the secretion of the antibody, the leader sequence of the bacterial enzyme pectate lyase was used. The leader sequence of this enzyme has already been used for the expression and secretion of a chimeric murine/human antibody (Fab fragment, Better et al., loc. cit.), and of the variable region of a humanized antibody (Ward et al., loc. cit.; Huse et al., loc. cit.). DNA for the first leader sequence (Px upstream of the heavy chain), and the sequence for a second ribosome binding site (RBS) and a second leader sequence (P2 upstream of the light chain) were synthesized from several oligonucleotides (Tab. 3).

Antibody cDNAs which code for the variable regions of the heavy and light chains of a human antibody (HuVhlys or HuVllys; Riechmann et al., (1988) J. Mol. Biol. 203, 825) were obtained from Dr. G. Winter (Cambridge, UK) . The restriction sites Hindlll (HuVhlys) and EcoRV (HuVllys) were introduced to make possible the insertion of the antibody cDNA into the expression vector. Further restriction sites for Banll (HuVhlys) and BstEII or Kpnl (HuVllys) were introduced to exchange hypervariable regions en bloc. At the end of the HuVhlys cDNA sequence a stop signal was incorporated. A Banll site in the light chain was removed. These alterations were carried out by means of site directed mutagenesis in the bacteriophage M13mpl8 (Zoller and Smith, Meth. Enzymol. 100, 468-500).

The sequence of the completed antibody DNA is shown in Tab. 4.

IE 91336 - 7 . For the insertion of the leader sequence P2 (Tab. 3) the modified plasmid pKK233-2 was digested using the restriction enzymes Ncol and Pstl, and Px was inserted in between these sites (pKK233-2-Px). Further cloning steps, apart from the last step, were carried out using the plasmid pUC18. The reason is that the presence of individual parts of the antibody operon in the expression vector adversely influences the growth of the bacterial host. 1Θ Before the cloning in pUC18, its BamHI restriction site had to be removed. After digesting with BamHI, the single-stranded ends were filled in using the Klenow fragment and were religated. This modified plasmid was then digested using Pstl and Hindlll, and P2 plus RBS was ligated in between the restriction sites (pUC18-P2).

During this process, the original Hindlll restriction site of the plasmid disappears and a new Hindlll restriction site is incorporated. pUC18-P2 was then digested using pstl and Hindlll, and the DNA of the heavy chain (Pstl-HindiII insert from Ml3) was ligated into these two sites (pUC18-HP2). This plasmid was then digested using EcoRV and BamHI, and the DNA of the light chain (EcoRVBamHI insert from M13) was ligated in (pUC18-HP2L).

The Pstl-BamHI insert was then recloned in pUC18 after the restriction sites for Hindlll, Banll and Kpnl therein had previously been removed. The Hindlll restriction site was removed as above for pKK233-2, the religation taking place without an insertion of BamHI linkers, however. Subsequently, the resulting plasmid was digested using Smal and Banll, and, after filling in the protruding ends by means of T4 DNA polymerase, religated. The insertion of the Pstl-BamHI restriction fragment results in pUCHP2L. In a preferred embodiment, a Tag sequence was additionally inserted in the Banll and Hindlll restriction sites (Tab. 3). The Tag sequence encodes the recognition sequence Glu-Gly-Glu-Glu-Phe of the monoclonal antibody Y1 1/2 (Wehland et al., (1984), EMBO J. 3, 1295). Because IE 91336 - 8 of this peptide marker the expression product of the resulting plasmid pUC-HTP2L is readily detectable.

For the insertion of HP2L or HTP2L in the expression vector, the two plasmids were cut using Pstl and BamHI, and the Pstl-BamHI HP2L insert from pUC-HP2L or the HTP2L insert from pUC-HTP2L was ligated into the modified plasmid pKK233-2-Pi into these two restriction sites. A diagrammatic representation of the completed expression vector pFMT is shown in Tab. 5.

Example 2: Synthesis of antibody DNA containing random sequences in hypervariable regions The synthesized oligonucleotides for the synthesis of the variable parts of antibody DNA are compiled in Tab. 1. For the synthesis of the hypervariable regions almost random nucleotide sequences were used. Limitations on the random nature are illustrated in Tab. 1. Two different sets of oligonucleotides were synthesized. In set A the hypervariable regions are predominantly random apart from those few positions where almost exclusively certain amino acids occur. In set B, the random nature of the nucleotide sequences in those positions where predominantly one or few amino acids occur was additionally limited.

The oligonucleotides were purified by HPLC chromatography or polyacrylamide gel electrophoresis, and then 5'-phosphorylated.

Example 3: Ligation of the synthetic oligonucleotides The oligonucleotides in Tab. 1 were ligated together stepwise on an antibody DNA template. For this purpose, large amounts (about 1 mg) of single-stranded M13mp=18 DNA containing the antibody DNA inserts were isolated. In order to separate the antibody DNA from the vector, the inserts were made double-stranded on the two ends using IE 91336 - 9 two appropriate oligonucleotides and were digested using the enzymes Pstl and Hindlll (heavy chain) or using EcoRV and BamHI (light chain). The antibody DNA was then purified using agar gel electrophoresis.

On these DNA templates, first only three oligonucleotides were ligated: Hl, pH2 and pH3 (heavy chain), and LI, pL2 and pL3 (light chain), Hl and LI having been marked first with 32P at their 5' end (p designates 5'-phosphorylated). Amounts of lOOpmol of each oligonucleotide were used. The hydridized oligonucleotides were purified on 2% agarose gels and analyzed on a sequencing gel. The amount was determined by a radioactivity measurement. Equimolar amounts of pH4 and pH5 (heavy chain), and pL4 and pL5 (light chain) were then ligated onto the already ligated oligonucleotides on each particular template. These DNAs were then purified as in the preceding step and the procedure was repeated up to the purification step, using equimolar amounts of pH6 and pH7. Finally, the ligated oligonucleotides were purified by means of a denaturing polyacrylamide gel and preferably amplified using the polymerase chain reaction (PCR). Alternatively or in order to avoid losses caused by the last purification step, the oligonucleotides were amplified using PCR directly after the last ligation step. The primers Hl and H8 (heavy chain), and LI and L6 (light chain) were used under standard conditions for the PCR. Amplified template DNA was digested selectively using Kpnl (light chain) or using Alul (heavy chain). Where appropriate, a second amplification step using the PCR was subsequently carried out.

Example 4: Insertion of the antibody DNA into the expression plasmid The synthesized antibody DNA was cut using the restriction enzymes Pstl and Banlll (heavy chain), and BstEII and Kpnl (light chain). The bands having the expected molecular weight were purified by agar gel 91336 - 10 electrophoresis, precipitated using ethanol and then, in two steps (first the DNA of the light chain and then the DNA of the heavy chain), ligated into the pUC-HP2L (see above) which had been cut and purified in the same way.

The HP2L insert was then ligated into the restriction sites Pstl and BamHI of the plasmids PKK233-2-P! (see Example 1). An analogous way was used for the HTP2L fragment. The antibody library is therefore established in the antibody expression plasmid (Tab. 6). The reason for intermediate cloning in pUC is that the presence of individual parts of the antibody operon in the expression vector has an adverse influence on the growth of the bacterial host (see above also).

Example 5: Expression and screening of antibodies in E. coli Competent E. coli are transfected with pFMT plasmids containing the inserted antibody-DNA library, grown on agarose plates and then incubated using nitrocellulose filters coated with the desired antigen. After removing non-specifically bound antibodies, the active clones are identified with a labeled antibody against the human immunoglobulins secreted from E. coli. In the preferred embodiment, the antibody YL 1/2 which is directed against the Tag sequence is used for this purpose.

IE 91336 ------- 11 , Legend for Fig. 1: Restriction map of the expression vector pKK233-2 (Amann and Brosius, loc. cit.).

Ptrc denotes hybrid tryptophan lac promoter RBS denotes ribosome binding site rrnB denotes ribosomal RNA B operon 5S denotes gene for 5S RNA Before cloning antibody DNA in the expression vector, the following alterations were carried out: 1) The Sail and EcoRI restriction sites were removed together with the DNA between them. 2) The Hindlll restriction site was converted to a BamHI restriction site.

IE 91336 - 12 TAB. 1 Oligonucleotide for the synthesis of a library of anti body DNA (variable parts) Set A Hl 5'CCAGGTCCAACTGCAGGAGAGCGGTCCAGGTCTTGTGAGACCTAG3 B2 H3 S' CCAGACCCTGAGCCTGACCTGCACCOTG3 ' 'TGTCTGGCTTCACCTTCAGC|Tl/2 TT| CTTT1/2TTTGGGTGCGCCAGCCACCTGGAC3 c C |C CC| A cc cc |A AG| G AA AG |G G |3 GG G H4 'GAGGTCTTGAGTGGATTGGT|T1/2TT| ΤΑΤ|Τ1/2ΤΤ| T1/2TACGCGTGACAATGCTGGTAGAC3' |C |A l<3 CC| |c CC| c c AG j |A AG j A A G j 10 |G G js 0 6 H5 H6 ' ACCAGCAAGAACCAGTTCAGCCTGCGTCTCAGCAGCGTGACAGC3' H7 HS LI L2 'CGCCGACACCGCGGTCTACTACTCTGCGCGC | Tl/2 TT | TGGGGTCAGGGCT3' |C CC| |A AG | |G 6 j10 'CCCTCGTCACAGTCTCCTCA3' ' CTGTGACGAGGCTGCCCTGACCCCA3' 'GCGCCAGCGTGGGTGACAGG3' ' GTGACCATCACCTGTT1/2TTGTT | T1/2TT | CTTT1/2TTTGGTAACAGCAGAAGCCAGGT3 ' C CC cc|c CC| A CC CC A A6 GA|A AG| G AA AG 6 6 G G G |7 GG G L3 L5 L6 'AAGGCTCCAAAGCTGCTGATCTAC | T1/2TT | GGTGTGCCAAGCCGTTTCAGCGGTAGCGGT3' |C CC| |A AG| |G G |7 'AGCGGTACGGACTTCACCTTCACCATCAGCAGCCTCCAGCCAGAGGAC3' 'ATCGCCACCTACTACTGCCAG | ΤΙ /2TT | TTCGGCCAAGGTAC3' |C CC| ,A AG j |G G |8 'CCACCTTGGTACCTTGGCCGAA3' IE 91336 Set B Hl, H2, H5, H7, H8 and LI, L4 and L6 are identical to those in set A.

H3 5'TGTCTGGCTTCACCTTCAGC AC10»T95« TT20«C TC20«G T5% TA20% T2« Τ5» Τ70» C C GA45«A5% CIO» GG80% Α85» G80« C28«A75»G30« G45« Α70» G10« A50«G20« G20« TGGGTGCGCCAGCCACCTGGAC3' H4 5'GAGGTCTTGAGTGGATTGGT T14.5«TT AT90«C T14.5»TT Τ5» T10»T70« T14.5*TT C28.5*CC GIO» C28.5*CC C7O»C8O«G3O« C28.5«CC A28.5«AG A28.5«AG A20«A10« A28.54AG 028.5«g G28.5«G 05« G28.5»G AAT A15«A20«C ACT AT16«A A70«C10«C TAX A80»C20»C CCC AC10»C T40«TC C5« Τ5» G GG G85«G60« GA C80« G30«A70«A G20«A60% GAA A40«A C20« A90«A90« G A4« G208 G20« 050« G40« G5« G5« A20«A10«T CGCGTGACAATGCTGGTAGAC3' G80»G90% E6 5' CGCCGACACCGCGGTCTACTACTGTGCGCGC Tl/2 TT GC25«C TATTGGGGTCAGGGCT3' C CC Α75» A AG G G8 L2 ' GTGACCAXCACCTGT CAA T30«CG AGT C75«AA T30«T10«C C10»T40«T90« AG G70« Α10» C104C30» A50»C10«A10« G15« Α304Α60» 640«A10« G30« G40« A70«T20»T70« C5« T5« T90« T90»T10«C C70«TA20« AC40«T TGGTAACAGCAGAAGCCAGGT3' G30»A40«A30« A90»C20«A10« C2« C5« Α20» G80« GA40* G40« G54.A40» A6« A85« G10« G20« G35% G2% L3 5'AAGGCTCCAAAGCTGCTGATCTAC T14.5»TT A40«T20«T AC5« C AC20»T70« CTA C28.54CC G60»C70« A10* A60«A30« G A28.5«AG A7« 085« G20« G28.5«G G3« C20%C70«C20« T70«CT GGTGTGCCAAGCCGTTTCAGCGGTAGCGGT3' G80«A30%A80« C15« Α15» X>5 'ATCGCCACCTACTACTGC CT10«A CT20»T10« T60»C30»G A90* A80»G90« A10«G70« G30« T35»T5« T C5« C20« A40«A50« G20«G25« T15»T5« C C10»C20* A60«A75« G15« T14.54TT CT15«C90« T14.5«TT ACGTTCGGCCAAGGTAC C28.54CC C70«A10% C28.54CC A28.54AG Α15» A28.5«AG G28.54G G28.54G IE 91336 - 14 Notes for Tab. 1 The random nature of the oligonucleotides of set B was limited in a manner which generates approximately the relevant amount of frequent amino acids in each position of the hupervariable regions (in accordance with the tables of Kabat et al, loc. cit.). In this strategy the number of expected stop codons was also reduced even further. In contrast with the oligonucleotides in set A, a restriction site for Mlul was not introduced.

IE 91336 - 15 (N CONSTRUCTION OF THE VECTOR pFMT FOR THE EXPRESSION AND SECRETION OF ANTIBODIES IN BACTERIA ft Eh H cn 0 2 HI D 2 cn H H CQ ω Q h 2 ft o O 0 cn ft O H EH EH ft H S ft D ft PA ft ft g Eh EH N o cn >H 41 0 ft ft HI Q ft O a ft S* EH s HI ft ft ft CO ►< cn s ft 41 ft o ω ft - w =* EH =* < H Eh z M cn U ft s 2 ft o O H ft Q Eh O W a H ft ft 0 λ 2 EH 2 cn BA ft H ft ft O § ft ft cn ft o ft ft EH 2 O » 9 ft H EH η O 0 41 $ ft o ft o ft Q ft EH Eh 2 ft H O cn l-H cn h ft Eh cn O l-H ω ω w ft ft X w H ft w Ε· I o Eh =» P/O: promoter/operator, RBS: ribosome binding site, P2: leader sequence of pectate lyase variable domain of the heavy chain, VL: variable domain of the light chain IE 91336 TAB. 3 Sequences of the leader sequences Pl and P2 in the antibody operon, and of the Tag sequences Pl Leader sequence of pectate lyase (Pl) MKYLLPTAAAGLLLLAAQPAMAQVQLQ CATGAAATACCTCTTGCCTACGGCAGCCGCTGGCTTGCT6CTGCTGGCAGCTCAGCCGGC6ATGGCGCAAGTTCAG£TGCAiGl Pstl P2 RBS Leader sequence of pectate lyase (P2) MKYLLPTAAA ί C1TGCAGCCAAGCTTGAATTCATTAAAGAGGAGAAATTAACTCCATGAAGTACTTACTGCCGACCGCTGCGGCG Pstl Hindlll GLLLLAAQPAMADI GGTCTCCTGCTGTTGGCGGCTCAGCCGGCTATGGCTGATATCGGATCCAGCT EcoRV BamHI The nucleotides in parentheses are the adjacent nucleotides of the plasmid The leader sequences were synthesized by hybridization of the following oligonucleotides.

Pl a. 5'CATGAAATACCTCTTGCCTACGGCAGCCGCTGGCTTG3' b. 5 ’ TTAACTCCATGAAGTACTTACTGCCGACCGCTGCG3 ’ c. 3’ACGTCGGTTCGAACTTAAGTTTTAACTCCTCTTTAATTGAGGTACTTCATGAATGACGGCTGGCGACGCCGCCCAGAGGA CGACAACCGCCGAGTCGGCC6ATACCGACTATAGCCTAGGTCGA5' d. 5'GCTCAGCCGGCTATGGCTGATATCGGATCC3' e. 5‘GCGGGTCTCCTGCTGTTGGCG3' The Tag sequences were synthesized by hybridization of the following sequences: a. 5' CCTTAGTCACAGTATCCTCAGAAGGTGAAGAATTCTA3' b. 5'AGCTTAGAATTCTTCACCTTCTGAGGATACTGTGACTAAGGAGCC3’ IE 91336 TAB. 4 Nucleotide sequences of antibody DNA a) Heavy chain (variable domain), HuVhlys Hindlll .........6 VHSQVQL QESGPGLVR CTCTCCACAGGTGTCCACTCCCAGGTCCAACTGCAGGAGAGCGGTCCAGGTCTTGTGAGA Pstl CDR1 PSQTLSLTCTVSGFTFS /6//Y//G/ CCTAGCCAGACCCTGAGCCTGACCTGCACCGTGTCTGGCTTCACCTTCAGCGGCTATGGT BspMI /V /N /V V R Q Ρ P G R G L E W I G /H/ 1/ W/ G/ GTAAACTGGGTGAGACAGCCACCTGGACGAGGTCTTGAGTGGATTGGAATGATTTGGGGT CDR2 60 70 /D /G /N /T /D /Y /N /S /A /L /K /S R V T M L V 0 T GATGGAAACACAGACTATAATTCAGCTCTCAAATCCAGAGTGACAATGCTGGTAGACACC - 90 SKNQFSLRLSSVTAADTAVY AGCAAGAACCAGTTCAGCCTGAGACTCAGCAGCGTGACAGCCGCCGACACCGCGGTCTAT Sacl I 100 CDR3 110 Y C A R E /R /0 /Y /R /L /D /Y W G Q G S L V T TATTGTGCAAGAGAGAGAGATTATAGGCTTGACTACTGGGGTCAGGGCTCCCTCGTCACA Ban 11 V S S Stop GTCTCCTCATAAGCTTCCTTACAACCTCTCTCTTCTATTCAGCTTAA.......BamHI HindHI b) Light chain (variable domain), Hu Vllys Hindlll GVHSDIQNTQSPSSLSA CTCTCCACAGGTGTCCACTCCGATATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCC EcoRV CORI 30 S V G D R V T I T C R/ A/ S/ G/ N/ 1/ Η/ N/ Y/ L AGCGTGGGTGACAGGGTGACCATCACCTGTAGAGCCAGCGGTAACATCCACAACTACCTG BstEII 50 CDR2 /A/ V Y 0 0 K P G K A Ρ K L L I Y /Y/ T/ T/ T GCTTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCTGCTGATCTACTACACCACCACC 70 /L/A/DGVPSRFSGSGSGTDFTF CTGGCTGACGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCTTC 90 CDR3 T I S S L Q Ρ E D I A T Y Y C /Q /H /F /V /S ACCATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCAGCACTTCTGGAGC 100 /T /P /R /T F G Q G T K V Ε I K R..ESTOP ACCCCAAGGACGTTCGGCCAAGGTACCAAGGTGGAAATCAAACGTGAGTAGAATTTAAAC Kpni TTTGCTTCCTCAGTTGGATCC BamHI ®91336 - 18 CM I CO co CM ft Ό Ό O ε IH urea The Antibody Expression Plasmid pFMT iudx IIHisa ΛΗ033 HIP"!H nuea HHSS5 n=*s DC «a IWdsg I red e c ο -H Ή (0 o»a φ υ n φ a -l O' a ή <0 M •H Μ Φ (Q a > 4-1 u Φ Ό (fl Φ co β CSS c o Ο -H •H (0 O'43 Φ U n Φ ι—I <0 a φ a a H Ll Φ « a > a Φ Ό (0 Φ CM ι co co CM ft Ό Φ M-l There is an RBS in the plasmid upstream of the heavy chain part but is not drawn in here.

IE 91336 ft Insertion of the antibody libraries in the expression vector pFMT CH t n n CH Ό Φ MH Ό i c c Ο 'rt rt (0 tji£ Φ O rt p Φ 43 —ι 0> Λ -rt Φ rH rt rt Φ (0 43 _> prt Φ •a td Φ co CQ e£ c c 0 -rt •rt Φ »43 Φ υ rt φ rrt β 4) Φ a) 43 •rt rt Φ a 43 > P rt Φ Ό «0 Φ CH I m n CH Cm •o Φ MH •rt Ό Ο ε IE 91336 - 20 - HOE 90/B 006J - Ma 834

Claims (16)

1. . Patent claims;

1. A synthetic human antibody library obtainable by generating random sequences for the hypervariable regions with the limitations (a) to (e), set A or set B according 5 to Tab. 1, and subsequent incorporation in an expression vector.

2. A synthetic human antibody library as claimed in claim 1, wherein the generated random sequences for the hypervariable regions are amplified before the incorporation 10 in an expression vector.

3. A synthetic human antibody library as claimed in claim 1 or 2, wherein the modified vectors Ml3mp18HuVhlys and M13mpl8HuVllys are used.

4. A synthetic antibody library as claimed in claim 1 to 15 3, wherein the vector pFMT is used as expression vector.

5. A process for preparing a synthetic human antibody library, which comprises generating random sequences for the hypervariable regions with the limitations (a) to (e), set A or set B according to Tab. 1, and then incor20 porating them in an expression vector.

6. The process as claimed in claim 5, wherein the generated random sequences are amplified before the incorporation in an expression vector.

7. The process as claimed in claim 5 or 6, wherein the 25 vector pFMT is used as expression vector.

8. A process for isolating clones which secrete specific human antibodies, which comprises screening synthetic human antibody libraries as claimed in claim 1 to 4 using specific antigens. 30

9. The process as claimed in claim 8., wherein a marker IE 91336 peptide, preferably the TAG sequence, is additionally incorporated and the desired clones are identified using antibodies against the marker peptide, preferably using the antibody YL 1/2. 5

10. The use of a synthetic human antibody library as claimed in claim 1, 2 or 3 for isolating clones which secrete specific antibodies.

11. A synthetic human antibody library according to claim 1, substantially as hereinbefore described and exemplified.

12. A process according to claim 5, for preparing a synthetic human antibody library, substantially as hereinbefore described and exemplified.

13. A synthetic human antibody library, whenever prepared by a process claimed in any one of claims 5-7 of 12.

14. A process according to claim 8 for isolating clones which secrete specific human antibodies, substantially as hereinbefore dexcribed and exemplified.

15. Clones which secrete specific human antibodies, whenever', isolated by a process claimed in ’any. one of claims 8, 9 or 14.

16. Use according to claim 10, substantially as hereinbefore described and exemplified.